More than 50 years ago, Sabin and Schlesinger adapted the wild type dengue type 1 virus (DEN1) and dengue type 2 virus (DEN2) to mice by serial intracerebral passage. This process rapidly selected for mouse neurovirulent mutants that concomitantly exhibited attenuation for susceptible humans. Later, attenuation of the DEN1 mouse neurovirulent mutant for humans was confirmed by scientists at the Walter Reed Army Institute of Research, who performed a large clinical trial with Sabin's original mouse neurovirulent mutant that had undergone a limited number of additional passages in mice. In addition, attenuation of another independently derived DEN2 mouse neurovirulent mutant was demonstrated by Schlesinger. Because of these provocative observations we sought to: (1) identify the mutations that are responsible for mouse neurovirulence in these and other mouse-adapted dengue virus mutants; and (2) identify the mutations in these mutants that might be responsible for attenuation for humans. Our sequence and biological analysis indicated that mouse-adapted DEN2 and DEN4 mutants contained multiple amino acid substitutions in the structural proteins and that one or more such changes were responsible for dengue mouse neurovirulence dependent on dengue virus serotype. Although the genetic loci of mouse neurovirulence appear to be different among dengue virus serotypes, it is possible to confer neurovirulence upon a non-neurovirulent dengue virus by introducing a mutation in the envelope protein (E) that is responsible for neurovirulence of a mouse-adapted neurovirulent mutant of another serotype. Mouse neurovirulent DEN4 (strain H241) replicated slowly and grew to a low titer in simian LLC-MK2 cells, whereas the non-neurovirulent parental virus grew rapidly to a high titer in the same cells. Analysis of virion proteins indicated that neurovirulent DEN4 H241 had a defect in virus maturation resulting from restriction of cleavage of PreM that is required to release mature M. The results of our genetic analysis indicated that reduction of PreM cleavage could be brought about by multiple substitutions in E or independently by one or two amino acid substitutions in PreM. We also investigated the genetic basis for growth-restriction of a mouse neurovirulent DEN1 vaccine (called MD-1) in simian cells. Similar to the situation identified for DEN4, growth restriction was caused by a single amino acid substitution near the PreM cleavage junction or independently by multiple amino acid changes in E. It was further noted that none of the PreM mutations in the DEN1 or DEN4 mutant played a role in mouse neurovirulence. Because dengue viruses are inherently not neurotropic, it is likely that mutations affecting viral growth, probably play a more important role in conferring attenuation on dengue neurovirulent mutants than mutations that affect only neurovirulence. For this reason, it might be desirable to construct a series of dengue virus mutants that contain two or more such growth restriction mutations for evaluation as candidate vaccine strains. Our goal in this project has been to identify the most promising attenuating mutations in order to set priorities for future studies of mutants in monkeys and ultimately, in adult volunteers.